CN106153876A - A kind of method utilizing blood glucose meter detection by quantitative biological marker - Google Patents
A kind of method utilizing blood glucose meter detection by quantitative biological marker Download PDFInfo
- Publication number
- CN106153876A CN106153876A CN201610804250.XA CN201610804250A CN106153876A CN 106153876 A CN106153876 A CN 106153876A CN 201610804250 A CN201610804250 A CN 201610804250A CN 106153876 A CN106153876 A CN 106153876A
- Authority
- CN
- China
- Prior art keywords
- agent
- capture
- biological marker
- complex
- saccharase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a kind of method that non-diseases diagnostic purpose utilizes blood glucose meter detection by quantitative biological marker, comprise the following steps: 1) capture biological marker, the method for described capture includes competing prize law or non-competing prize law；2) by described step 1) signal of biological marker that captures is converted into glucose signals；3) glucose signals that blood glucose meter detection converts is utilized.Biological marker signal is converted into glucose signals by the present invention, and uses blood glucose meter as detecting instrument, it is possible to simple and quick detection by quantitative purpose biological marker, and result can pass through blood glucose meter Digital output, the shortest, low cost.
Description
Technical field
The present invention relates to biomolecule detection field, particularly relate to a kind of blood glucose meter detection by quantitative biological marker of utilizing
Method.
Background technology
Detection method about biological marker has electrophoresis method, radioassay method, traditional fluorescent spectrometry, immunity
Method, chromatography etc., the most also rise a lot of molecular biology for detection based on PCR, although these methods relatively pass
System method accuracy is good, highly sensitive, but these detection methods need to use the specific equipment of laboratory, need the skill of specialty
Art personnel carry out detection operation, time-consuming long and cost height.Therefore, be not suitable for the quickly detection of biological marker and identify.
Summary of the invention
In view of this, a kind of method that it is an object of the invention to provide fast and convenient detection biological marker.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides a kind of method that non-diseases diagnostic purpose utilizes blood glucose meter detection by quantitative biological marker, including
Following steps: 1) capture biological marker, the method for described capture includes competing prize law or non-competing prize law；2) by described
Step 1) signal of biological marker that captures is converted into glucose signals；3) the glucose letter that blood glucose meter detection converts is utilized
Number；
Described step 1) in non-competing prize law specifically include following steps:
1.1) capture agent of active substrate is connected by the biology in non-competing catching method capture testing sample
Marker, obtains substrate-capture agent-biological marker complex；
1.2) by identification agent and the described step 1.1 of activity) substrate-capture agent-biological marker of obtaining is combined
Thing mixes 30～60min at 20～30 DEG C, obtains substrate-capture agent-biological marker-identification agent complex；
1.3) by described step 1.2) substrate-capture agent-biological marker-identification agent complex of obtaining be connected
Thing Hybrid connections, obtains substrate-capture agent-biological marker-identification agent-junctional complex complex；
Described step 2) specifically include following steps:
2.1) by described step 1.3) substrate-capture agent-biological marker-identification agent-junctional complex of obtaining is combined
It is compound that thing and the saccharase Hybrid connections of activity obtain substrate-capture agent-biological marker-identification agent-junctional complex-enzyme
Thing；
2.2) by sucrose solution and substrate-capture agent-biological marker-identification agent-junctional complex-multienzyme complex 20
～after 30 DEG C of mixing 1～3h, the signal of biological marker is converted into the mixed liquor to be measured of glucose signals；
Described step 3) be: use blood glucose meter to detect the glucose signals in described mixed liquor to be measured.
Preferably, described step 1) use the catching method capture biological marker of competition to specifically include following steps:
A) active matrix is connected with capture agent, obtains connecting the capture agent of active substrate；
B) capture agent of active substrate will be connected and obtain substrate-catch with being connected the identification agent connection having saccharase
Obtain reagent-connection and have the identification agent complex of saccharase；
C) the identification agent complex of saccharase there is is to mix with testing sample substrate-capture agent-connection, testing sample
In biological marker be connected the identification agent competition binding capture agent having saccharase so that connect and have the identification of saccharase
Reagent has disengaging the identification agent complex of saccharase from substrate-capture agent-connection, obtains substrate-capture agent-biology
Marker complex.
Preferably, after using competition prize law capture biological marker, described step 2) detection of glucose signals is right
As the connection for departing from has the identification agent of saccharase；
Described step 3) be: detected described glucose signals by blood glucose meter.
Preferably, described step B) in connection have the preparation method of identification agent of saccharase to comprise the following steps:
Saccharase is reacted 30～60min with coupling agent Hybrid connections, obtains coupling agent-saccharase complex；
The identification agent of sulfydryl modification, PB buffer and three (2-carboxyethyl) phosphine are mixed, stands 30～60min, obtain
The identification agent of activation；
Described coupling agent-saccharase complex is mixed with the identification agent of described activation, stands 24～48h, connected
It is connected to the identification agent of saccharase.
Preferably, the reaction temperature that described saccharase is connected with coupling agent mixing is 20～30 DEG C, and described mixing connects
It is to carry out under conditions of rotating speed is 20～30r/min.
Preferably, described step 1.1) in non-competing catching method specifically include following steps:
A) active matrix is connected with capture agent, obtains connecting the capture agent of active substrate；
B) testing sample connects with the capture agent being connected active substrate, obtains substrate-capture agent-biological marker
Complex.
Preferably, slide, carboxyl magnetic bead, Protein A magnetic bead or strepto-after described active matrix is activation are affine
Element-magnetic bead.
Preferably, described step 1.1) described in the capture agent connecting active substrate by comprising the following steps
Method prepares: active matrix and capture agent are 20～30 DEG C, coupling 1～2h under conditions of stirring, turning of described stirring
Speed is 20～30r/min.
Preferably, also including standing after described coupling, the temperature of described standing is 0～8 DEG C, and the time of described standing is
8～12h.
Preferably, described step 1.3) in junctional complex include for Streptavidin or coupling agent.
Preferably, described coupling agent include sulfo-SMCC, succinimido 3-[Bromoacetyl amino] propyl ester,
Succinimido 4-[p-maleimide phenyl] butyrate or succinimido-6-[(β-maleimide propionic acid amide.
Base)] own ester.
Preferably, described step 1.2) in the identification agent of activity be the identification examination that biotin or chemical group are modified
Agent.
Preferably, described step 2.1) in activity saccharase include biotinylated saccharase.
Preferably, step 1.3) described in capture agent-biological marker-identification agent complex and junctional complex mix
The time closing connection is 30～60min, and described Hybrid connections is carried out under conditions of rotating speed is 20～30r/min.
Preferably, step 2.1) described in capture agent-biological marker-identification agent-junctional complex complex with
The temperature of the saccharase Hybrid connections of activity is 20～30 DEG C, and the time is 30～60min.
Preferably, described biological marker is DNA, RNA, protein, biological micromolecule, cancerous cell, antibacterial and virus
In one.
Present invention also offers a kind of test kit for detecting biological marker, including capture biological marker reagent,
The signal conversion reagent of biological marker and auxiliary reagent；Described capture biological marker reagent includes connecting active substrate
Capture agent, activity identification agent and junctional complex；The signal conversion reagent of described biological marker includes the sucrose of activity
Enzyme and sucrose solution；Described auxiliary reagent includes Buffer solution A and morpholino b acid-polysorbas20 solution, described
Buffer solution A is PBST solution.
The signal of biological marker is converted into glucose signals by the method that the present invention provides, and uses blood glucose meter as inspection
Survey instrument, detect the amount of glucose by blood glucose meter thus obtain the amount of biological marker, it is possible to realize simple and quick detection
Purpose, and quantitative testing result can be obtained, result can pass through blood glucose meter Digital output, the shortest, low cost.
Accompanying drawing illustrates:
Fig. 1 is the schematic diagram utilizing blood glucose meter detection by quantitative biological marker；
Fig. 2 be embodiment 9 when junctional complex is Streptavidin, utilize showing of blood glucose meter detection by quantitative biological marker
It is intended to.
Fig. 3 is as a example by porcine circovirus 2 type PCV2, utilizes the schematic diagram of blood glucose meter detection by quantitative biological marker.
Detailed description of the invention
The invention provides a kind of method that non-diseases diagnostic purpose utilizes blood glucose meter detection by quantitative biological marker, including
Following steps: 1) capture biological marker, the method for described capture includes competing prize law or non-competing prize law；2) by described
Step 1) signal of biological marker that captures is converted into glucose signals；3) the glucose letter that blood glucose meter detection converts is utilized
Number；
Described step 1) in non-competing prize law specifically include following steps: 1.1) connect the capture agent of active substrate
By the biological marker in non-competing catching method capture testing sample, obtain substrate-capture agent-biological marker multiple
Compound；1.2) by identification agent and the described step 1.1 of activity) substrate-capture agent-biological marker complex of obtaining exists
20～30 DEG C of mixing 30～60min, obtain substrate-capture agent-biological marker-identification agent complex；1.3) by described
Step 1.2) substrate-capture agent-biological marker-identification agent complex of obtaining and junctional complex Hybrid connections, obtain base
Matter-capture agent-biological marker-identification agent-junctional complex complex；
Described step 2) specifically include following steps: 2.1) by described step 1.3) substrate-capture agent-biology of obtaining
Marker-identification agent-junctional complex complex obtains substrate-capture agent-biological label with the saccharase Hybrid connections of activity
Thing-identification agent-junctional complex-multienzyme complex；2.2) by sucrose solution and substrate-capture agent-biological marker-identification examination
The signal of biological marker, after 20～30 DEG C of mixing 1～3h, is converted into glucose signals by agent-junctional complex-multienzyme complex
Mixed liquor to be measured；Described step 3) be: use blood glucose meter to detect the glucose signals in described mixed liquor to be measured.
Fig. 1 is the schematic diagram utilizing blood glucose meter detection by quantitative biological marker.When using non-competing catching method capture
During biological marker, blood glucose meter detection glucose signals time, send glucose signals material be complex as shown in Figure 1
Overall；When using the catching method capture biological marker of competition, during blood glucose meter detection glucose signals, send glucose letter
Number the connection that is an off of material have the identification agent of saccharase, the i.e. identification agent of saccharase, junctional complex and the activity of activity
Complex.
The method that the present invention provides, preferably by the biological marker in double antibody sandwich method capture testing sample, this
The bright identification agent preferably by the capture agent and activity that connect active substrate captures biological marker jointly.In the present invention
In, the described capture agent connecting active substrate is the most relative with biological marker to be detected with the identification agent of activity
Should；
In the present invention, described biological marker is preferably DNA, RNA, protein, biological micromolecule, cancerous cell, antibacterial
With the one in virus；When biological marker is DNA, RNA, the capture agent of the active substrate of described connection is preferably with upper
Stating the capture probe that nucleic acid is corresponding, the identification agent of described activity is preferably the identification probe corresponding with above-mentioned nucleic acid；Work as biology
When marker is protein, cancerous cell, antibacterial and virus, the capture agent of the active substrate of described connection is preferably corresponding list
Clonal antibody, the identification agent of described activity is preferably corresponding polyclonal antibody；When biological marker is biological micromolecule,
The capture agent of the active substrate of described connection is preferably corresponding biotin-aptamers, and the identification agent of described activity is preferred
For the signal probe that be connected with saccharase complementary with aptamers, specifically, when biological marker is biological micromolecule, preferably adopt
Capturing biological marker with the catching method of competition, described connection has the identification agent of saccharase for complementary with aptamers
Probe, utilizing competition law to capture biological micromolecule, and biological micromolecule squeezing out and connecting the identification agent having saccharase
Time, just can directly collect the connection squeezed out has the identification agent of saccharase to carry out follow-up signal test experience.
In the present invention, described active matrix is preferably carboxyl magnetic bead or the ProteinA magnetic bead of activation, and described capture tries
Agent is preferably connected to the microsphere surface of carboxyl magnetic bead by covalent coupling, or described capture agent is preferably by the Fc of capture agent
Section is specific binding with ProteinA magnetic bead and is connected.
In the present invention, first will connect capture agent and the testing sample Hybrid connections of active substrate, obtain substrate-
Capture agent-biological marker complex.In the present invention, described substrate is commercial goods, when described substrate is carboxyl magnetic bead
Time, the concentration of described carboxyl magnetic bead is preferably 10mg/mL, the present invention before described carboxyl magnetic bead is connected with capture agent,
Preferably carrying out the activation of carboxyl magnetic bead, the activation of described carboxyl magnetic bead uses the activating means that this area is conventional, without other
Particular determination, in the embodiment of the present invention, the activation of carboxyl magnetic bead specifically includes following steps:
Carboxyl magnetic bead Magneto separate removes supernatant, and described Magneto separate uses the magnetic force test tube rack that this area is conventional, described
The carboxyl magnetic bead of Magneto separate is preferably disposed in centrifuge tube, and the specification of described centrifuge tube is preferably 1.5ml.
In the present invention, after completing carboxyl magnetic bead Magneto separate, use morpholino b acid-polysorbas20 solution washing carboxyl magnetic
Pearl, in described morpholino b acid-polysorbas20 solution, the concentration of morpholino b acid is preferably 100mM, described morpholino b acid-tell
Temperature 20 solution preferably also including, 0.05wt%Tween 20, described morpholino b acid-polysorbas20 solution obtain pH and be preferably
5.0, the volume of described morpholino b acid-polysorbas20 solution and the volume ratio of carboxyl magnetic bead are preferably 3～6: 2, preferred
For 2:1；The number of times of described washing is preferably 1～3 time, more preferably 2 times.The present invention preferably removes after washing every time
Clear liquid；
In the present invention, after completing the washing of carboxyl magnetic bead, preferably use activated solution that it is carried out the activation that suspends.This
Activated solution described in invention preferably comprises 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide solution and N-hydroxysuccinimidyl
The mixed solution of imide solution.In the present invention, described 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide solution
Concentration is preferably 10mg/mL, and the concentration of described N-hydroxy-succinamide solution is preferably 10mg/mL；Described 1-(3-bis-
Methylaminopropyl) volume ratio of-3-ethyl carbodiimide solution and N-hydroxy-succinamide solution is preferably 1:1.
In the present invention, the described activation that suspends is preferably: the carboxyl magnetic bead after described washing is placed in described activated solution
In suspend.The present invention does not has special restriction to the mode realizing suspending, and in an embodiment of the present invention, specifically can use
Rotating blending instrument makes carboxyl magnetic bead fully suspend.In the present invention, the described temperature suspending activation is preferably 22～28 DEG C, more excellent
Choosing for 25 DEG C；The described time suspending activation is preferably 30～60min, more preferably 40～50min；The described activation that suspends is excellent
Being selected in rotating speed is to carry out under conditions of 20～30rpm, more preferably 25rpm.
When described substrate is that it is activated by the method that ProteinA magnetic bead uses this area conventional, special without other
Different requirement.
The present invention, for the ease of the use of activated substrate, preferably carries out Magneto separate after the described activation that suspends, removes supernatant,
Obtain activated substrate.
The activated substrate obtained, after obtaining activated substrate, is connected by the present invention with capture agent, and described connection preferably has
Body comprises the steps:
Capture agent is mixed with activated substrate and carries out coupled reaction；In the present invention, described capture agent and activation base
The volume ratio of matter is preferably 1:1；The temperature of described coupled reaction is preferably 22～28 DEG C, more preferably 25 DEG C；Described
The time of coupled reaction is preferably 0.5～2h, more preferably 1h.Described coupled reaction is preferably suspended by the present invention in system
Carrying out under state, the present invention does not has a special restriction to the method keeping described suspended state, in an embodiment of the present invention, and can
Concrete employing rotates blending instrument and keeps system to be in suspended state；In the present invention, the rotating speed of described rotation blending instrument is preferred
It is 20～30rpm, more preferably 25rpm.
The reaction system obtained, after described connection terminates, is preferably stood, obtains being connected with catching of active matrix by the present invention
Obtain reagent.In the present invention, the temperature of described standing is preferably 0～4 DEG C；The time of described standing preferably 10～12h.
The present invention after obtaining connecting the capture agent of active substrate, preferably close on active matrix not with capture examination
The group of agent reaction.Currently preferred use contains the PBST solution (pH 7.2, containing 0.5%Tween-20) of BSA to described
On active matrix, unreacted radical is closed；In the present invention, the concentration of described PBST solution be preferably 0.01～
0.015mol/L；The time of described closing is preferably 1～2h；The temperature of described closing is preferably 22～28 DEG C, more preferably
For 25 DEG C.During described closing, the present invention preferably makes system keep suspended state；In an embodiment of the present invention,
The capture agent connecting active substrate can be kept to be in suspended state by specifically used rotation blending instrument, described rotation blending instrument
Rotating speed is preferably 20～30rpm, more preferably 25rpm.
It is preferably saved backup after obtaining being connected with the capture agent of active matrix by the present invention.In the present invention,
The described capture agent being connected with active matrix preferably preserves in the 0.01M PBST solution containing 1%BSA.Institute in the present invention
That states capture agent preserves the body preferably using the 0.01M PBST solution containing 1%BSA, described PBST solution and capture agent
Long-pending ratio preferably 2～4:1；In the present invention, the temperature of described preservation is preferably 0～4 DEG C.
In the present invention, the identification agent of described activity is mixed to get with substrate-capture agent-biological marker complex
Substrate-capture agent-biological marker-identification agent complex, the temperature of described mixing is preferably 20～30 DEG C, more preferably
25 DEG C, the time of described mixing is preferably 30～60min, more preferably 40～50min.
The identification agent of heretofore described activity is preferably biotin or the identification agent of chemical group modification.Described
Biotin be preferably commercially available Sulfo-NHS-LC-Biotin, use described biotin Fresh biotin solution.This
The concrete steps that biotin and identification agent connect are included by invention: by molten to identification agent and above-mentioned freshly prepared biotin
Liquid mix, carry out mixing and hatch the identification agent obtaining biotin modification, the concentration of described identification agent be preferably 0.8～
1.2mg/ml, more preferably 1mg/ml, the volume of described identification agent is preferably 0.5～1ml, described biotin solution
Concentration is preferably 8～12mM, more preferably 10mM, and the volume of described biotin solution is preferred with the volume ratio of identification agent
For 1:50～150, more preferably 1:90.In the present invention, described in hatch and preferably hatch on ice or incubated at room,
The described time hatched on ice is preferably 1～3h, more preferably 2h；The temperature that described room temperature gives is preferably 20～30
DEG C, more preferably 25 DEG C, the time of described incubated at room is preferably 30～60min, more preferably 40～50min；This
Invention is after hatching end, and the mixed liquor after preferably hatching carries out ultrafiltration 3～removes unreacted biotin in mixed liquor 5 times.
In the present invention, those skilled in the art do not have special restriction to the identification agent that described chemical group is modified, and use ability
The commercially available prod of the identification agent that the chemical group known to field technique personnel is modified, such as Jin Sirui bio tech ltd
The nucleic probe of the sulfydryl modification provided.
The present invention, by substrate-capture agent-biological marker-identification agent complex and junctional complex Hybrid connections, obtains
Substrate-capture agent-biological marker-identification agent-junctional complex complex, described junctional complex is preferably coupling agent or strepto-parent
And element, described coupling agent is preferably sulfo-SMCC；The concentration of described Streptavidin is preferably 1～3 μM, preferred
Being 2 μMs, preferred described Streptavidin is excellent with the volume ratio of substrate-capture agent-biological marker-identification agent complex
Elect 1:1 as, described substrate-capture agent-biological marker-identification agent complex and Streptavidin Hybrid connections time
Between preferably 30～60min, more preferably 40～50min.Described substrate-capture agent-biological marker-identification examination
After agent complex and Streptavidin Hybrid connections terminate, again carry out Magneto separate, preferably wash 2～5 by Buffer solution A
Secondary.Described coupling agent sulfo-SMCC preferably sulfydryl with substrate-capture agent-biological marker-identification agent complex enters
Row connects.Described connection preferably particularly as follows: saccharase is connected 30～60min with coupling agent sulfo-SMCC mixing, obtains idol
Connection agent-saccharase complex；By substrate-capture agent-biological marker-identification agent complex and the coupling of sulfydryl modification
Agent-saccharase complex mixing, places 24～48h, obtains substrate-capture agent-biological marker-identification agent-junctional complex
Complex connects the identification agent having saccharase.Described sulfydryl is preferably modified on identification agent；In the present invention, identification agent
Activation preferably include following steps: the identification agent of sulfydryl modification, PB buffer and three (2-carboxyethyl) phosphine are mixed, place
30～60min, obtain the identification agent of activation.
The present invention is obtaining substrate-capture agent-biological marker-identification agent-junctional complex complex, by substrate-catch
Obtain reagent-biological marker-identification agent-junctional complex complex and obtain substrate-capture examination with active saccharase Hybrid connections
Agent-biological marker-identification agent-junctional complex-multienzyme complex；The saccharase of described activity is preferably biotinylated sucrose
Enzyme, the concentration of described biotinylated saccharase is preferably 0.4～0.8mg/mL, more preferably 0.6mg/mL, preferably
The volume of described biotinylation saccharase and the body of substrate-capture agent-biological marker-identification agent-junctional complex complex
Long-pending ratio is 1:1.Described substrate-capture agent-biological marker-identification agent-junctional complex complex and biotinylation sucrose
The temperature of enzyme Hybrid connections is preferably 20～30 DEG C, more preferably 25 DEG C, and the time is preferably 30～60min, more preferably
For 40～50min.The present invention is obtaining substrate-capture agent-biological marker-identification agent-junctional complex-multienzyme complex
After, the most again carry out Magneto separate, and wash 2～5 times by Buffer solution A.As a example by PCV2 virus, heretofore described
The structure of monoclonal antibody-virus-how anti-Streptavidin-multienzyme complex as shown in Figure of description 3, connect and have carboxyl magnetic bead
After PCV2 monoclonal antibody and biotinylation PCV2 multi-resistance capture PVC2 jointly, it is connected with Streptavidin, biotinylation saccharase.
The present invention is after obtaining monoclonal antibody-virus-how anti-Streptavidin-multienzyme complex, by sucrose solution and substrate-catch
Obtain reagent-biological marker-identification agent-junctional complex-multienzyme complex to obtain by biology mark after 20～30 DEG C of mixing 1～3h
The signal knowing thing is converted into the mixed liquor to be measured of glucose signals；The time of described mixing is preferably 1～3h, more preferably
2h；The concentration of described sucrose solution is preferably 0.2～1M, more preferably 0.5M；Preferably, described sucrose solution is with to be measured
The volume ratio of mixed liquor is 1:1, and described sucrose solution is that sucrose is dissolved in Buffer A and is prepared from.
The present invention, after obtaining mixed liquor to be measured, uses blood glucose meter to detect the glucose signals in mixed liquor to be measured, according to
The amount of the glucose that blood glucose meter detects thus calculate the amount of virus.
Described step 1.1) in non-competing catching method specifically include following steps:
A) active matrix is connected with capture agent, obtains connecting the capture agent of active substrate；
B) testing sample connects with the capture agent being connected active substrate, obtains substrate-capture agent-biological marker
Complex.
In the present invention, slide, carboxyl magnetic bead, Protein A magnetic bead or the strepto-parent after described active matrix is activation
With element-magnetic bead.
In the present invention, described step 1.1) described in connect active substrate capture agent by comprising the following steps
Method prepare: active matrix and capture agent 20～30 DEG C, coupling 1～2h under conditions of stirring, described stirring
Rotating speed is 20～30r/min.
In the present invention, also including standing after described coupling, the temperature of described standing is 0～8 DEG C, the time of described standing
It is 8～12h.
In the present invention, described step 1.3) in junctional complex include Streptavidin or coupling agent.
In the present invention, described coupling agent preferably includes sulfo-SMCC, succinimido 3-[Bromoacetyl ammonia
Base] propyl ester, succinimido 4-[p-maleimide phenyl] butyrate, succinimido-6-[(β-maleimide
Propionamido-)] own ester.
In the present invention, described step 1.2) in the identification agent of activity be the identification that biotin or chemical group are modified
Reagent.
In the present invention, described step 2.1) in activity saccharase include biotinylated saccharase.
In the present invention, step 1.3) described in capture agent-biological marker-identification agent complex and junctional complex
The time of Hybrid connections is preferably 30～60min, and described Hybrid connections preferably enters under conditions of rotating speed is 20～30r/min
OK.
In the present invention, step 2.1) described in capture agent-biological marker-identification agent-junctional complex complex
Being preferably 20～30 DEG C with the temperature of the saccharase Hybrid connections of activity, the time is preferably 30～60min.
In the present invention, described step 1) use the catching method capture biological marker of competition to specifically include following step
Rapid:
A) active matrix is connected with capture agent, obtains connecting the capture agent of active substrate；
B) capture agent of active substrate will be connected and obtain substrate-catch with being connected the identification agent connection having saccharase
Obtain reagent-connection and have the identification agent complex of saccharase；
C) the identification agent complex of saccharase there is is to mix with testing sample substrate-capture agent-connection, testing sample
In biological marker be connected the identification agent competition binding capture agent having saccharase so that connect and have the identification of saccharase
Reagent has disengaging the identification agent complex of saccharase from substrate-capture agent-connection, obtains substrate-capture agent-biology
Marker complex.
In the present invention, described step B) in connection have the preparation method of identification agent of saccharase to include following step
Rapid:
Saccharase is reacted 30～60min with coupling agent Hybrid connections, obtains coupling agent-saccharase complex；
The identification agent of sulfydryl modification, PB buffer and three (2-carboxyethyl) phosphine are mixed, stands 30～60min, obtain
The identification agent of activation；
Described coupling agent-saccharase complex is mixed with the identification agent of described activation, stands 24～48h, connected
It is connected to the identification agent of saccharase.
In the present invention, the reaction temperature that saccharase is connected with coupling agent mixing is preferably 20～30 DEG C, and described mixing is even
Connect and preferably carry out under conditions of rotating speed is 20～30r/min.
In the present invention, described step B) in connection have the preparation method of identification agent of saccharase to include following step
Rapid: with coupling agent mixing, saccharase is connected 30～60min, more preferably 45min, and described connection temperature is preferably 20～30
DEG C, more preferably 25 DEG C, described mixing connection is preferably carried out under conditions of rotating speed is 20～30r/min, more preferably 25r/
Min, obtains coupling agent-saccharase complex；The identification agent of sulfydryl modification, PB buffer and three (2-carboxyethyl) phosphine is mixed
Close, place 30～60min, more preferably 40min, obtain the identification agent of activation；By coupling agent-saccharase complex and activation
Identification agent mixing, place 24～48h, more preferably 36h, obtain connecting the identification agent having saccharase.
The solvent of the buffer of described preparation testing sample is preferably Buffer B solution, described Buffer B solution
Being preferably PBST solution, pH 7.2, containing 0.5%Tween-20 and 1g/L BSA.
After described Hybrid connections obtains substrate-capture agent-biological marker complex, preferably carry out Magneto separate, use
Buffer solution A is washed 2～5 times, and described Buffer solution A is preferably PBST solution, and pH 7.2, containing 0.5%Tween-
20 and 1g/L BSA.
Present invention also offers a kind of test kit for detecting biological marker, including capture biological marker reagent,
The signal conversion reagent of biological marker and auxiliary reagent；Described capture biological marker reagent includes connecting active substrate
Capture agent, activity identification agent and junctional complex；The signal conversion reagent of described biological marker includes the sucrose of activity
Enzyme and sucrose solution；Described auxiliary reagent includes Buffer solution A and morpholino b acid-polysorbas20 solution, described
Buffer solution A is PBST solution.
The method of blood glucose meter detection by quantitative biological marker is utilized to carry out in detail offer of the present invention below in conjunction with embodiment
Thin explanation, but they can not be interpreted as limiting the scope of the present invention.
Embodiment 1
By carboxyl magnetic bead and the connection of porcine circovirus 2 type PCV2 monoclonal antibody of 100 μ L activation, obtain connecting the carboxylic having monoclonal antibody
Base magnetic bead, then carries out Magneto separate, washes 4 times by Buffer solution A.After Magneto separate, carboxyl magnetic bead precipitation is scattered in 100 μ L to contain
There is the Buffer solution A of PCV2 Cap cap albumen then by rotating blending instrument at 25 DEG C, the condition of 25r/min
Under, mix 2h, then carry out Magneto separate carboxyl magnetic bead, wash 3 times by Buffer solution A.100 μ are added in carboxyl magnetic bead precipitates
L 1mg/L biotinylation PCV2 multi-resistance (being dissolved in Buffer solution A), then mixes 45min, Magneto separate carboxyl magnetic at 28 DEG C
Pearl, washes 4 times by Buffer solution A, carboxyl magnetic bead precipitation is scattered in 2 μMs of Streptavidins of 80 μ L and (is dissolved in Buffer A molten
Liquid) in, mix 50min.Magneto separate carboxyl magnetic bead again, washes 2 times by Buffer solution A, and carboxyl magnetic bead precipitation is scattered in 50 μ L
0.6mg/mL biotin-saccharase junctional complex (being dissolved in Buffer solution A) in, at 25 DEG C gently mix conjunction 40min, Magneto separate
And wash 5 times by Buffer solution A, take 65 μ L 0.5M sucrose solution (being dissolved in Buffer solution A) and join in carboxyl magnetic bead,
After 1h, take 5 μ L solution blood glucose meter and detect.
Embodiment 2
By carboxyl magnetic bead and the connection of porcine circovirus 2 type PCV2 monoclonal antibody of 60 μ L activation, obtain connecting the carboxylic having activation
Base magnetic bead, then carries out Magneto separate, washes 3 times by Buffer solution A.After Magneto separate, carboxyl magnetic bead precipitation is scattered in 100 μ L to contain
There is the Buffer solution A (containing 25% porcine blood serum) of PCV2 Cap cap albumen, then existed by rotation blending instrument
28 DEG C, under conditions of 24r/min, mix 2h, then carry out Magneto separate carboxyl magnetic bead, wash 3 times by Buffer solution A.At carboxyl
Magnetic bead precipitation adds 55 μ L 1mg/L biotinylation PCV2 multi-resistance (being dissolved in Buffer solution A), then mixes at 28 DEG C
55min, Magneto separate carboxyl magnetic bead, wash 2 times by Buffer solution A, magnetic bead precipitation is scattered in 2 μMs of Streptavidins of 60 μ L
In (being dissolved in Buffer solution A), mix 45min.Magneto separate carboxyl magnetic bead again, washes 4 times by Buffer solution A, carboxyl magnetic
Pearl precipitation is scattered in the 0.6mg/mL biotin-saccharase junctional complex (being dissolved in Buffer solution A) of 50 μ L, gently stirs at 25 DEG C
Mixing 40min, Magneto separate is also washed 5 times by Buffer solution A, takes 65 μ L 0.5M sucrose solution (being dissolved in Buffer solution A) and adds
Enter in magnetic bead, after 3h, take 5 μ L solution blood glucose meter and detect.
Embodiment 3
By the connection of the ProteinA magnetic bead of 80 μ L activation with porcine circovirus 2 type PCV2 monoclonal antibody, obtaining connection has activation
ProteinA magnetic bead, then carry out Magneto separate, wash 2 times by Buffer solution A.After Magneto separate, ProteinA magnetic bead is precipitated
It is scattered in Buffer A (containing the 25% porcine blood serum) solution that 55 μ L contain PCV2 Cap cap albumen, then passes through
Rotation blending instrument is at 25 DEG C, under conditions of 28r/min, mixes 2h, then carries out Magneto separate ProteinA magnetic bead, use Buffer A
Solution washes 3 times.In magnetic bead precipitates, add 55 μ L 1mg/L biotinylation PCV2 multi-resistance (being dissolved in Buffer solution A), then exist
Mix 50min, Magneto separate magnetic bead at 28 DEG C, wash 2 times by Buffer solution A, ProteinA magnetic bead precipitation is scattered in 60 μ L
In 1.8 μMs of Streptavidins (being dissolved in Buffer solution A), mix 45min.Magneto separate ProteinA magnetic bead, uses Buffer again
Solution A is washed 4 times, and ProteinA magnetic bead precipitation is scattered in the 0.6mg/mL biotin-saccharase junctional complex of 50 μ L and (is dissolved in Buffer
Solution A) in, gently mix conjunction 40min at 25 DEG C, Magneto separate is also washed 5 times by Buffer solution A, is taken 65 μ L 0.5M sucrose solutions
(being dissolved in Buffer solution A) joins in ProteinA magnetic bead, after 3h, takes 5 μ L solution blood glucose meter and detects.
Embodiment 4
60 μ L Streptavidins-magnetic bead is connected with the Biotin-capture probe 1 of RNA to be measured, obtains connecting capture probe
Streptavidin-the magnetic bead of 1, then carries out Magneto separate, washes 3 times by Buffer solution A.By Streptavidin-magnetic after Magneto separate
Pearl precipitation is scattered in Buffer solution A that 100 μ L contain RNA to be measured then by rotating blending instrument at 28 DEG C, 24r/min's
Under the conditions of, mix 2h, then carry out Magneto separate Streptavidin-magnetic bead, wash 3 times by Buffer solution A.Streptavidin-
Magnetic bead precipitation adds the Biotin-capture probe 2 (being dissolved in Buffer solution A) of 50 μ L RNA to be measured, then mixed at 28 DEG C
Close 60min, Magneto separate Streptavidin-magnetic bead, wash 2 times by Buffer solution A, magnetic bead precipitation is scattered in 2 μMs of chains of 60 μ L
In mould Avidin (being dissolved in Buffer solution A), mix 45min.Magneto separate Streptavidin-magnetic bead, molten with Buffer A again
Liquid is washed 4 times, and Streptavidin-magnetic bead precipitation is scattered in the 0.6mg/mL biotin-saccharase junctional complex of 50 μ L and (is dissolved in Buffer
Solution A) in, gently mix conjunction 40min at 25 DEG C, Magneto separate is also washed 5 times by Buffer solution A, is taken 65 μ L 0.5M sucrose solutions
(being dissolved in Buffer solution A) joins in magnetic bead, after 3h, takes 5 μ L solution blood glucose meter and detects.
Embodiment 5
The carboxyl magnetic bead of 100 μ L activation is connected with the monoclonal antibody of testing protein, obtains connecting the carboxyl magnetic bead having monoclonal antibody,
Then carry out Magneto separate, wash 4 times by Buffer solution A.After Magneto separate, carboxyl magnetic bead precipitation is scattered in 100 μ L and contains to be measured
The Buffer solution A of protein then by rotate blending instrument at 25 DEG C, under conditions of 25r/min, mix 2h, then carry out
Magneto separate carboxyl magnetic bead, washes 3 times by Buffer solution A.In carboxyl magnetic bead precipitates, add 100 μ L 1mg/L biotinylations treat
Survey the multi-resistance (being dissolved in Buffer solution A) of protein, at 28 DEG C, then mix 45min, Magneto separate carboxyl magnetic bead, use
Buffer solution A is washed 4 times, carboxyl magnetic bead precipitation is scattered in 2 μMs of Streptavidins of 80 μ L (being dissolved in Buffer solution A),
Mixing 50min.Magneto separate carboxyl magnetic bead again, washes 2 times by Buffer solution A, and carboxyl magnetic bead precipitation is scattered in 50 μ L's
In 0.6mg/mL biotin-saccharase junctional complex (being dissolved in Buffer solution A), gently mixing conjunction 40min at 25 DEG C, Magneto separate is also
Wash 5 times by Buffer solution A, take 65 μ L 0.5M sucrose solution (being dissolved in Buffer solution A) and join in carboxyl magnetic bead, 1h
After, take 5 μ L solution blood glucose meter and detect.
Embodiment 6
The carboxyl magnetic bead of 100 μ L activation is connected with the monoclonal antibody of cell to be measured, obtains connecting the carboxyl magnetic bead having monoclonal antibody, so
After carry out Magneto separate, wash 4 times by Buffer solution A.After Magneto separate by carboxyl magnetic bead precipitation be scattered in 100 μ L contain to be measured carefully
The Buffer solution A of born of the same parents then by rotate blending instrument at 25 DEG C, under conditions of 25r/min, mix 2h, then carry out magnetic and divide
From carboxyl magnetic bead, wash 3 times by Buffer solution A.100 μ L 1mg/L biotinylations are added to be measured carefully in carboxyl magnetic bead precipitates
The multi-resistance (being dissolved in Buffer solution A) of born of the same parents, then mixing 45min at 28 DEG C, Magneto separate carboxyl magnetic bead, molten with Buffer A
Liquid is washed 4 times, carboxyl magnetic bead precipitation is scattered in 2 μMs of Streptavidins of 80 μ L (being dissolved in Buffer solution A), mixes 50min.
Magneto separate carboxyl magnetic bead again, washes 2 times by Buffer solution A, and the 0.6mg/mL that carboxyl magnetic bead precipitation is scattered in 50 μ L is biological
In element-saccharase junctional complex (being dissolved in Buffer solution A), gently mixing conjunction 40min at 25 DEG C, Magneto separate is the most molten with Buffer A
Liquid is washed 5 times, takes 65 μ L 0.5M sucrose solution (being dissolved in Buffer solution A) and joins in carboxyl magnetic bead, after 1h, takes 5 μ L solution
Detect by blood glucose meter.
Embodiment 7
60 μ L Streptavidins-magnetic bead is connected with the Biotin-capture probe 1 of nucleus to be measured, obtains connection and catch
Obtain the Streptavidin-magnetic bead of probe 1, then carry out Magneto separate, wash 3 times by Buffer solution A.By strepto-parent after Magneto separate
It is scattered in Buffer solution A that 100 μ L contain nucleus to be measured then by rotating blending instrument 28 with element-magnetic bead precipitation
DEG C, under conditions of 24r/min, mix 2h, then carry out Magneto separate Streptavidin-magnetic bead, wash 3 times by Buffer solution A.
The Biotin-capture probe 2 adding 50 μ L nucleus to be measured in Streptavidin-magnetic bead precipitation (is dissolved in Buffer A molten
Liquid), at 28 DEG C, then mix 60min, Magneto separate Streptavidin-magnetic bead, wash 2 times by Buffer solution A, magnetic bead is sunk
Shallow lake is scattered in 2 μMs of Streptavidins of 60 μ L (being dissolved in Buffer solution A), mixes 45min.Magneto separate strepto-is affine again
Element-magnetic bead, washes 4 times by Buffer solution A, and Streptavidin-magnetic bead precipitation is scattered in the 0.6mg/mL biotin-sugarcane of 50 μ L
In carbohydrase junctional complex (being dissolved in Buffer solution A), gently mixing conjunction 40min at 25 DEG C, Magneto separate also washes 5 by Buffer solution A
Secondary, take 65 μ L 0.5M sucrose solution (being dissolved in Buffer solution A) and join in magnetic bead, after 3h, take 5 μ L solution blood glucose meter and examine
Survey.
Embodiment 8
The carboxyl magnetic bead of 100 μ L activation is connected with the monoclonal antibody of tested bacteria, obtains connecting the carboxyl magnetic bead having monoclonal antibody, so
After carry out Magneto separate, wash 4 times by Buffer solution A.After Magneto separate by carboxyl magnetic bead precipitation be scattered in 100 μ L contain to be measured carefully
The Buffer solution A of bacterium then by rotate blending instrument at 25 DEG C, under conditions of 25r/min, mix 2h, then carry out magnetic and divide
From carboxyl magnetic bead, wash 3 times by Buffer solution A.100 μ L 1mg/L biotinylations are added to be measured carefully in carboxyl magnetic bead precipitates
The multi-resistance (being dissolved in Buffer solution A) of bacterium, then mixing 45min at 28 DEG C, Magneto separate carboxyl magnetic bead, molten with Buffer A
Liquid is washed 4 times, carboxyl magnetic bead precipitation is scattered in 2 μMs of Streptavidins of 80 μ L (being dissolved in Buffer solution A), mixes 50min.
Magneto separate carboxyl magnetic bead again, washes 2 times by Buffer solution A, and the 0.6mg/mL that carboxyl magnetic bead precipitation is scattered in 50 μ L is biological
In element-saccharase junctional complex (being dissolved in Buffer solution A), gently mixing conjunction 40min at 25 DEG C, Magneto separate is the most molten with Buffer A
Liquid is washed 5 times, takes 65 μ L 0.5M sucrose solution (being dissolved in Buffer solution A) and joins in carboxyl magnetic bead, after 1h, takes 5 μ L solution
Detect by blood glucose meter.
Embodiment 9
60 μ L Streptavidins-magnetic bead is connected with the Biotin-capture probe 1 of tested bacteria nucleic acid, obtains connection and catch
Obtain the Streptavidin-magnetic bead of probe 1, then carry out Magneto separate, wash 3 times by Buffer solution A.By strepto-parent after Magneto separate
It is scattered in Buffer solution A that 100 μ L contain tested bacteria nucleic acid then by rotating blending instrument 28 with element-magnetic bead precipitation
DEG C, under conditions of 24r/min, mix 2h, then carry out Magneto separate Streptavidin-magnetic bead, wash 3 times by Buffer solution A.
The Biotin-capture probe 2 adding 50 μ L tested bacteria nucleic acid in Streptavidin-magnetic bead precipitation (is dissolved in Buffer A molten
Liquid), at 28 DEG C, then mix 60min, Magneto separate Streptavidin-magnetic bead, wash 2 times by Buffer solution A, magnetic bead is sunk
Shallow lake is scattered in 2 μMs of Streptavidins of 60 μ L (being dissolved in Buffer solution A), mixes 45min.Magneto separate strepto-is affine again
Element-magnetic bead, washes 4 times by Buffer solution A, and Streptavidin-magnetic bead precipitation is scattered in the 0.6mg/mL biotin-sugarcane of 50 μ L
In carbohydrase junctional complex (being dissolved in Buffer solution A), gently mixing conjunction 40min at 25 DEG C, Magneto separate also washes 5 by Buffer solution A
Secondary, take 65 μ L 0.5M sucrose solution (being dissolved in Buffer solution A) and join in magnetic bead, after 3h, take 5 μ L solution blood glucose meter and examine
Surveying, Fig. 2 is the schematic diagram that embodiment 9 utilizes blood glucose meter detection by quantitative biological marker.
Embodiment 10
60 μ L Streptavidins-magnetic bead is connected with the Biotin-aptamers of biological micromolecule to be measured, obtains connecting adaptation
Streptavidin-the magnetic bead of body, then carries out Magneto separate, washes 3 times by Buffer solution A.In Streptavidin-magnetic bead precipitation
The Invertase-signal probe (being dissolved in Buffer solution A) that middle addition 50 μ L is complementary with aptamers, then mixes at 28 DEG C
60min, Magneto separate Streptavidin-magnetic bead, wash 2 times by Buffer solution A, by Streptavidin-magnetic bead precipitation after Magneto separate
It is scattered in Buffer solution A that 100 μ L contain biological micromolecule to be measured then by rotating blending instrument at 28 DEG C, 24r/min's
Under the conditions of, mix 2h, then carry out Magneto separate Streptavidin-magnetic bead, collect supernatant.Take 100 μ L 0.5M sucrose solutions
(being dissolved in Buffer solution A) joins in supernatant, after 3h, takes 5 μ L solution blood glucose meter and detects.
Embodiment 11
Take 400 μ L 2mg/mL saccharase (being dissolved in BufferA solution) and add 1mg sulfo-SMCC coupling agent, mixing, shake
Swing 1h, be centrifuged off insoluble sulfo-SMCC, then with BufferA solution ultrafiltration 8 times, obtain coupling agent-saccharase and be combined
Thing.Take the capture probe 2 (being dissolved in ultra-pure water) of the sulfydryl modification of 30 100 μMs of RNA to be measured of μ L, add 10 μ L 0.2M PB
(pH5.5) and 2 μ L 50mM TCEP (being dissolved in ultra-pure water), mixing, room temperature is placed 1h, with BufferA solution ultrafiltration 8 times, is obtained
The capture probe 2 of activation.Being mixed homogeneously with the capture probe 2 of activation by coupling agent-saccharase complex, room temperature places 48h, uses
BufferA solution ultrafiltration 8 times, obtains capture probe 2-saccharase complex.By 60 μ L Streptavidins-magnetic bead and RNA to be measured
Biotin-capture probe 1 connect, obtain connect capture probe 1 Streptavidin-magnetic bead, then carry out Magneto separate, use
Buffer solution A washes 3 times.After Magneto separate, Streptavidin-magnetic bead precipitation is scattered in the Buffer that 100 μ L contain RNA to be measured
Solution A then by rotate blending instrument at 28 DEG C, under conditions of 24r/min, mix 2h, then carry out Magneto separate strepto-affine
Element-magnetic bead, washes 3 times by Buffer solution A.50 μ L capture probe 2-saccharases are added multiple in Streptavidin-magnetic bead precipitation
Compound, then mixes 60min at 28 DEG C, and Magneto separate is also washed 5 times by Buffer solution A, taken 65 μ L 0.5M sucrose solutions (molten
In Buffer solution A) join in magnetic bead, after 3h, take 5 μ L solution blood glucose meter and detect.
Embodiment 12
A kind of test kit detecting biological marker use, as a example by detecting virus, forms including with lower part, capture disease
Poison reagent, the signal conversion reagent of virus, auxiliary reagent；Described capture virus agent includes connecting the capture having carboxyl magnetic bead
Reagent, the identification agent of activity, Streptavidin；The signal conversion reagent of described virus includes biotinylation saccharase, sugarcane
Sugar juice；Described auxiliary reagent is the reagent used in virus detection procedure, including Buffer A and morpholino b acid-tween
20 solution；Described Buffer solution A is PBST solution, and the pH of Buffer solution A is 7.2, contains in Buffer solution A
0.5%Tween-20 and 1g/L BSA；The concentration of described morpholino b acid-polysorbas20 solution is 100mM morpholino b acid,
The pH 5.0 of quinoline ethyl sulfonic acid-polysorbas20 solution, described morpholino b acid-polysorbas20 solution contains the Tween 20 of 0.05%.
As seen from the above embodiment, the method for detection biological marker of the present invention is simple and quick, and passes through blood
Sugar instrument detects the amount of glucose thus obtains the amount of biological marker, it is possible to detection by quantitative purpose biological marker, result can be led to
Cross blood glucose meter Digital output, the shortest, low cost.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (17)
1. the method that a non-diseases diagnostic purpose utilizes blood glucose meter detection by quantitative biological marker, it is characterised in that include with
Lower step: 1) capture biological marker, the method for described capture includes competing prize law or non-competing prize law；2) by described step
Rapid 1) signal of the biological marker captured is converted into glucose signals；3) the glucose letter that blood glucose meter detection converts is utilized
Number；
Described step 1) in non-competing prize law specifically include following steps:
1.1) capture agent of active substrate is connected by the biological label in non-competing catching method capture testing sample
Thing, obtains substrate-capture agent-biological marker complex；
1.2) by identification agent and the described step 1.1 of activity) substrate-capture agent-biological marker complex of obtaining exists
20～30 DEG C of mixing 30～60min, obtain substrate-capture agent-biological marker-identification agent complex；
1.3) by described step 1.2) substrate-capture agent-biological marker-identification agent complex of obtaining mixes with junctional complex
Close and connect, obtain substrate-capture agent-biological marker-identification agent-junctional complex complex；
Described step 2) specifically include following steps:
2.1) by described step 1.3) substrate-capture agent-biological marker-identification agent-junctional complex complex of obtaining with
The saccharase Hybrid connections of activity obtains substrate-capture agent-biological marker-identification agent-junctional complex-multienzyme complex；
2.2) by sucrose solution and substrate-capture agent-biological marker-identification agent-junctional complex-multienzyme complex 20～30
DEG C mixing 1～3h after, the signal of biological marker is converted into the mixed liquor to be measured of glucose signals；
Described step 3) be: use blood glucose meter to detect the glucose signals in described mixed liquor to be measured.
Method the most according to claim 1, it is characterised in that described step 1) use the catching method of competition to capture biology
Marker specifically includes following steps:
A) active matrix is connected with capture agent, obtains connecting the capture agent of active substrate；
B) capture agent of active substrate will be connected and obtain substrate-capture examination with being connected the identification agent connection having saccharase
Agent-connection has the identification agent complex of saccharase；
C) the identification agent complex of saccharase there is is to mix with testing sample, in testing sample substrate-capture agent-connection
Biological marker be connected the identification agent competition binding capture agent having saccharase so that connect and have the identification agent of saccharase
Have the identification agent complex of saccharase from substrate-capture agent-connection and depart from, obtain substrate-capture agent-biological label
Thing complex.
Method the most according to claim 2, it is characterised in that after using competition prize law capture biological marker, described
Step 2) the detection object of glucose signals is that the connection departed from has the identification agent of saccharase；
Described step 3) be: detected described glucose signals by blood glucose meter.
Method the most according to claim 2, it is characterised in that described step B) in connection have the identification agent of saccharase
Preparation method comprise the following steps:
Saccharase is reacted 30～60min with coupling agent Hybrid connections, obtains coupling agent-saccharase complex；
The identification agent of sulfydryl modification, PB buffer and three (2-carboxyethyl) phosphine are mixed, stands 30～60min, activated
Identification agent；
Being mixed with the identification agent of described activation by described coupling agent-saccharase complex, stand 24～48h, obtaining connection has
The identification agent of saccharase.
Method the most according to claim 4, it is characterised in that described saccharase mixes, with coupling agent, the reaction temperature being connected
Being 20～30 DEG C, described mixing connection is to carry out under conditions of rotating speed is 20～30r/min.
Method the most according to claim 1, it is characterised in that described step 1.1) in non-competing catching method specifically wrap
Include following steps:
A) active matrix is connected with capture agent, obtains connecting the capture agent of active substrate；
B) testing sample connects with the capture agent being connected active substrate, obtains substrate-capture agent-biological marker and is combined
Thing.
Method the most according to claim 1, it is characterised in that described active matrix be activation after slide, carboxyl magnetic bead,
ProteinA magnetic bead or Streptavidin-magnetic bead.
Method the most according to claim 1, it is characterised in that described step 1.1) described in connect active substrate
Capture agent is prepared by the method comprised the following steps: active matrix and capture agent are 20～30 DEG C, the condition of stirring
Lower coupling 1～2h, the rotating speed of described stirring is 20～30r/min.
Method the most according to claim 8, it is characterised in that also include after described coupling standing, the temperature of described standing
Being 0～8 DEG C, the time of described standing is 8～12h.
Method the most according to claim 1, it is characterised in that described step 1.3) in junctional complex include for strepto-parent
With element or coupling agent.
11. methods according to claim 10, it is characterised in that described coupling agent includes that sulfo-SMCC, succinyl are sub-
Amido 3-[Bromoacetyl amino] propyl ester, succinimido 4-[p-maleimide phenyl] butyrate or butanimide
Base-6-[(β-maleimide propionamido-)] own ester.
12. methods according to claim 1, it is characterised in that described step 1.2) in activity identification agent make a living
The identification agent that thing element or chemical group are modified.
13. methods according to claim 1, it is characterised in that described step 2.1) in the saccharase of activity include raw
The saccharase of thing element.
14. methods according to claim 1, it is characterised in that step 1.3) described in capture agent-biological label
The time of thing-identification agent complex and junctional complex Hybrid connections is 30～60min, described Hybrid connections rotating speed be 20～
Carry out under conditions of 30r/min.
15. methods according to claim 1, it is characterised in that step 2.1) described in capture agent-biological label
Thing-identification agent-junctional complex complex is 20～30 DEG C with the temperature of saccharase Hybrid connections of activity, the time be 30～
60min。
16. methods according to claim 1, it is characterised in that described biological marker is DNA, RNA, protein, biology
One in little molecule, cancerous cell, antibacterial and virus.
17. 1 kinds of test kits being used for detecting biological marker, including capture biological marker reagent, the signal of biological marker
Conversion reagent and auxiliary reagent；Described capture biological marker reagent includes connecting the capture agent of active substrate, activity
Identification agent and junctional complex；The signal conversion reagent of described biological marker includes saccharase and the sucrose solution of activity；Institute
The auxiliary reagent stated includes BufferA solution and morpholino b acid-polysorbas20 solution, and described BufferA solution is that PBST is molten
Liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610804250.XA CN106153876A (en) | 2016-09-06 | 2016-09-06 | A kind of method utilizing blood glucose meter detection by quantitative biological marker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610804250.XA CN106153876A (en) | 2016-09-06 | 2016-09-06 | A kind of method utilizing blood glucose meter detection by quantitative biological marker |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106153876A true CN106153876A (en) | 2016-11-23 |
Family
ID=57340996
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610804250.XA Pending CN106153876A (en) | 2016-09-06 | 2016-09-06 | A kind of method utilizing blood glucose meter detection by quantitative biological marker |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106153876A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107664631A (en) * | 2017-07-25 | 2018-02-06 | 刘斐 | The preparation of equipment, detection method and its sample based on smart mobile phone detection biological marker |
| CN108303541A (en) * | 2017-01-11 | 2018-07-20 | 上海鸣捷生物科技有限公司 | A kind of porcine circovirus type 2 antibody testing kit and its detection method |
| CN111424115A (en) * | 2020-03-13 | 2020-07-17 | 南京农业大学 | Method for detecting novel coronavirus by using glucometer |
| CN112322698A (en) * | 2020-10-22 | 2021-02-05 | 温州医科大学 | Bacterium detection method and kit by utilizing nano silver-sucrose invertase compound |
| CN113759108A (en) * | 2021-09-07 | 2021-12-07 | 南昌航空大学 | Method for measuring content of chloramphenicol by biosensor based on glucometer |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103025885A (en) * | 2010-05-26 | 2013-04-03 | 伊利诺伊大学评议会 | Personal glucose meters for detection and quantification of a broad range of analytes |
| CN103353521A (en) * | 2013-06-04 | 2013-10-16 | 华中师范大学 | Method for determining contents of biological marker and DNA through direct-reading portable glucometer |
-
2016
- 2016-09-06 CN CN201610804250.XA patent/CN106153876A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103025885A (en) * | 2010-05-26 | 2013-04-03 | 伊利诺伊大学评议会 | Personal glucose meters for detection and quantification of a broad range of analytes |
| CN103353521A (en) * | 2013-06-04 | 2013-10-16 | 华中师范大学 | Method for determining contents of biological marker and DNA through direct-reading portable glucometer |
Non-Patent Citations (4)
| Title |
|---|
| QING WANG ET AL: "Sensitivepoint-of-caremonitoringofcardiacbiomarkermyoglobin using aptamerandubiquitouspersonalglucosemeter", 《BIOSENSORS ANDBIOELECTRONICS》 * |
| TIAN LAN ET AL: "Detection of Protein Biomarker Using a Blood Glucose Meter", 《METHODS MOL BIOL》 * |
| YU XIANG ET AL: "Using the Widely Available Blood Glucose Meter to Monitor Insulin and HbA1c", 《JOURNAL OF DIABETES SCIENCE AND TECHNOLOGY》 * |
| 谭贵良等: "《现代分子生物学及组学技术在食品安全检测中的应用》", 30 June 2014 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108303541A (en) * | 2017-01-11 | 2018-07-20 | 上海鸣捷生物科技有限公司 | A kind of porcine circovirus type 2 antibody testing kit and its detection method |
| CN108303541B (en) * | 2017-01-11 | 2023-07-25 | 上海鸣捷生物科技有限公司 | Porcine circovirus type 2 antibody detection kit and detection method thereof |
| CN107664631A (en) * | 2017-07-25 | 2018-02-06 | 刘斐 | The preparation of equipment, detection method and its sample based on smart mobile phone detection biological marker |
| CN107664631B (en) * | 2017-07-25 | 2024-02-13 | 南京农业大学 | Device and method for detecting biological marker based on smart phone and preparation of sample thereof |
| CN111424115A (en) * | 2020-03-13 | 2020-07-17 | 南京农业大学 | Method for detecting novel coronavirus by using glucometer |
| CN112322698A (en) * | 2020-10-22 | 2021-02-05 | 温州医科大学 | Bacterium detection method and kit by utilizing nano silver-sucrose invertase compound |
| CN112322698B (en) * | 2020-10-22 | 2021-10-15 | 温州医科大学 | Bacterium detection method and kit by utilizing nano silver-sucrose invertase compound |
| CN113759108A (en) * | 2021-09-07 | 2021-12-07 | 南昌航空大学 | Method for measuring content of chloramphenicol by biosensor based on glucometer |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106153876A (en) | A kind of method utilizing blood glucose meter detection by quantitative biological marker | |
| CN101438163B (en) | Detection of target molecules in a sample by using a magnetic field | |
| CN104459157B (en) | A kind of preparation method of the immunosensor based on ferrocene-cuprous oxide cubic nanometer Framework Mark and application | |
| CN104391117B (en) | Preparation method and application of gastric cancer antigen electrogenerated chemiluminescence sensor based on PP<y>-NH2GO-Ag2Se@CdSe | |
| CN106018806B (en) | One kind is based on Au Fe3O4The preparation method and application for the interlayer type immunosensor that@Pd ZIF 8 are marked | |
| US20190070604A1 (en) | Systems and apparatus for detecting compounds in human biological samples | |
| CN102994638B (en) | Sandwich immuno-PCR (polymerase chain reaction) detection method and kit for to-be-detected object in biological samples | |
| CN111413264A (en) | Multi-channel particle detection device and method for detecting micron particles | |
| CN107328930A (en) | A kind of preparation and application based on dual signal response ratio type screen printing electrode immunosensor | |
| Yin et al. | A microfluidic chip-based MRS immunosensor for biomarker detection via enzyme-mediated nanoparticle assembly | |
| CN106970224A (en) | A kind of kit of application CD45 immunofluorescences joint CEP probe identification circulating tumor cells and its application | |
| CN103320503B (en) | Nanometer material probe system and detection method for tubercle bacillus detection | |
| DK2734842T3 (en) | Apparatus and method for laterale flow affinity assay | |
| Zhang et al. | Cluster-bomb type magnetic biosensor for ultrasensitive detection of Vibrio parahaemolyticus based on low field nuclear magnetic resonance | |
| CN109444240A (en) | A kind of electrochemistry immuno-sensing method established based on Prussian blue electrochemical immunosensor and based on the sensor and application | |
| CN103207218B (en) | Electrochemical immunosensor making method and Streptococcus suis detection method using electrochemical immunosensor | |
| CN206038688U (en) | Immunity chromatography detection test strip's fluorescent quantitation spectral detection system | |
| CN111398236A (en) | Micro-fluidic chip detection system, application and antigen-antibody immunodetection method | |
| CN101551391A (en) | Immuomagnetic bead chromatographic test strip for rapidly detecting chloromycetin and preparation method thereof | |
| Keller et al. | On-slide detection of enzymatic activities in selected single cells | |
| US20200200742A1 (en) | Method of detecting specimen substance using multiphase polymer fine particles | |
| CN115728495A (en) | Charge-mediated micro-channel current biosensing method based on directional coupling technology and application thereof | |
| GB2591009A (en) | Composite target-tumor serum nucleic acid ligand detection method and kit | |
| CN109682964B (en) | Au@Fe3O4MNPs-Ab2Preparation method of nano enzyme detection probe and method for detecting multi-component antigen | |
| CN104034898A (en) | Kit for quantitatively detecting CEA (Carcino Embryonie Antigen) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Liu Fei Inventor after: Zhang Yan Inventor after: Zhou Jiyong Inventor after: Xing Gang Inventor before: Liu Fei Inventor before: Zhang Yan |
|
| COR | Change of bibliographic data | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20161123 |
|
| RJ01 | Rejection of invention patent application after publication |